The ability to manipulate and engineer genomic sequences has helped to elucidate basic biological processes, enhance the production of biochemicals and pharmaceuticals, and provide insight into clinically relevant disease states. The disruption or deletion of genes has been used to identify essential genes in a variety of organisms and to investigate the functions of unknown genes. While much has been learned from random deletions, it is desirable to target a specific gene or region of a chromosome for deletion. Commonly used methods for deleting genes or chromosomal regions include homologous recombination and site-specific recombination. Homologous recombination has the disadvantage that extensive regions of sequence similarity are required between the recombining DNA sequences. To date, deletion experiments using a site-specific recombination system (such as Cre/loxP and Flp/FRT) are cumbersome in that they require the construction of several customized targeting constructs or complex PCR experiments.
Attempts have been made to circumvent some of these difficulties. FRT recombination sites have been inserted permanently at random locations in the E. coli genome using a transposon (Tn5) (U.S. Pat. No. 6,140,129). Similarly, Tn5 transposons were used to insert loxP recombination sites at random locations in E. coli, and the region between the sites was deleted by expression of Cre recombinase (Yu et al. 2002, Nature Biotechnology 20, 1018-1023). Transposons insert at random locations in the DNA, however. Thus, transposon-mediated insertions of recombination sites do not permit precise or targeted deletions. In another attempt, loxP recombination sites were positioned at specific locations in the Corynebacterium glutamicum genome by homologous recombination using strain-specific island sequences (Suzuki et al. 2005, Applied Environmental Microbiol. 71, 3369-3372). This method is restricted for use in bacterial genomes, and in only those with strain-specific islands. A need exists, therefore, for a simple method to position site-specific recombination sites at targeted locations in a broad range of organisms, whereby the region between the recombination sites at the targeted locations may be deleted by a site-specific recombinase.